Systematic calorimetric studies of proton exchange associated with binding of beta-lactoglobulin with ligand

In proteins, proton exchange is caused due to the changes in the proton affinity (pK_a) of ionizable groups that are engaged in conformational changes induced by the binding of a ligand. In addition, knowledge regarding the type and number of such ionizable groups is very important to understand the pH-dependent changes of the thermodynamic parameters. Therefore, in this study, we performed a comprehensive analysis of proton exchange by using beta lactoglobulin (Blg), a representative of the lipocalin family of proteins. We used isothermal titration calorimetry to evaluate the proton exchange during binding with sodium dodecyl sulfate (SDS) at different pHs ranging from 2 to 9. SDS binds to Blg in all studied range of pHs and enthalpy-driven reactions are observed in acidic pH, whereas enthalpy-entropy driven reactions are observed in neutral and basic pHs. Enthalpy-entropy compensation leads to relatively small changes in the association constants with the maximal value at pH = 8.0. The simultaneous analysis of the number of exchanged protons, the binding constants, and the enthalpy was performed in the range pH 5.5-9. At least 4 residues that change their ionization pattern are needed to explain the observed pH dependence.